The eukaryotic cytoskeleton is critical for cell migration. How the dynamic microtubule and actin cytoskeletal networks interact to coordinate polarized cell migration is poorly understood. Spectraplakins are key cytoskeletal cross-linkers that track microtubule plus ends and engage the leading edge actin network in migrating cells. Mutations in spectraplakins result in a wide spectrum of diseases including defective neuronal migration, brain malformations, neurodegeneration, and delayed wound healing. While the mechanism underlying spectraplakin binding to actin filaments is well established, how spectraplakins engage the microtubule cytoskeleton is poorly understood. Spectraplakins have a C-terminal EF-Hand-GAS2 microtubule- binding module and a proximal EB1-binding SxIP motif that confers microtubule plus end tracking. The architecture of the EF-Hand-GAS2 module, the identification of its microtubule-binding determinants, and an understanding of how the EF-Hand-GAS2-SxIP modules synergistically confer localization to leading edge microtubules remains to be determined. We hypothesize that the spectraplakin EF-Hand-GAS2 module forms a composite microtubule-binding structure that works synergistically with the EB1-binding SxIP motif to promote microtubule binding at the leading edge of migrating cells. Three series of experiments examine the structure, function and mechanism of the spectraplakin microtubule-binding module in cell migration. The first objective is to determine the atomic structure of the EF-Hand-GAS2 module using X-ray crystallography to elucidate how the EF-Hand and GAS2 domains collectively form a composite microtubule- binding structure. The second objective is to functionally map residues in the EF-Hand-GAS2 module involved in microtubule-binding and determine how the proximal EB1-binding SxIP module affects microtubule binding in vitro using microtubule dynamics reconstitution assays. The third objective is to characterize the synergistic behavior of the EF-Hand-GAS2 and SxIP modules in migrating cells. This investigation will use live cell and fixed cell fluorescence imaging to assay spectraplakin localization and cytoskeletal dynamics in motile cells. These three independent aims work to develop a multi-resolution model for spectraplakin microtubule-binding activity in migrating cells. The investigation's long term objective is to determine how spectraplakins regulate and coordinate microtubule and actin dynamics in neuronal growth cones. A mechanistic understanding of spectraplakins will enhance our knowledge of the inter-cytoskeletal coordination processes that underlie polarized neuronal migration and inform how spectraplakin mutations yield aberrant brain structure and neuronal connectivity. The proposed research will impact public health by establishing a mechanistic framework from which defective cell migration can be investigated, providing molecular insight into mutant spectraplakin phenotypes including delayed wound response, aberrant neuronal migration, and defective brain architecture.